Long noncoding RNA BCRP3 stimulates VPS34 and autophagy activities to promote protein homeostasis and cell survival

Abstract

Background Autophagy plays important roles in cell homeostasis and protein quality control. Long non-coding RNAs (lncRNAs) have been revealed as an emerging class of autophagy regulators, but the majority of them function in regulating the expression of autophagy-related genes. LncRNAs that directly act on the core autophagic proteins remain to be explored. Methods Immunofluorescence staining and Western blotting were used to evaluate the function of BCRP3 in autophagy and aggrephagy. RNA immunoprecipitation and in vitro RNA–protein binding assay were used to evaluate the interaction of BCRP3 with its target proteins. Phosphatidylinositol 3-phosphate ELISA assay was used to quantify the enzymatic activity of VPS34 complex. qRT-PCR analysis was used to determine BCRP3 expression under stresses, whereas mass spectrometry and Gene Ontology analyses were employed to evaluate the effect of BCRP3 deficiency on proteome changes. Results We identified lncRNA BCRP3 as a positive regulator of autophagy. BCRP3 was mainly localized in the cytoplasm and bound VPS34 complex to increase its enzymatic activity. In response to proteotoxicity induced by proteasome inhibition or oxidative stress, BCRP3 was upregulated to promote aggrephagy, thereby facilitating the clearance of ubiquitinated protein aggregates. Proteomics analysis revealed that BCRP3 deficiency under proteotoxicity resulted in a preferential accumulation of proteins acting in growth inhibition, cell death, apoptosis, and Smad signaling. Accordingly, BCRP3 deficiency in proteotoxic cells compromised cell proliferation and survival, which was mediated in part through the upregulation of TGF-β/Smad2 pathway. Conclusions Our study identifies BCRP3 as an RNA activator of the VPS34 complex and a key role of BCRP3-mediated aggrephagy in protein quality control and selective degradation of growth and survival inhibitors to maintain cell fitness.

AutophagyNet: High-resolution data source for the analysis of autophagy and its regulation

“…While initially it was thought that autophagy is solely regulated at the protein level, a seminal report in 1999 showed that gene regulatory changes affect autophagy at a transcriptional level [64]. Moreover, autophagy regulation on the post-transcriptional level by non-coding RNAs has also been described, but less studied [65][66][67]. Although the knowledge on autophagy regulation has been expanded significantly, data on post-translational and (post-)transcriptional regulations are scattered across many resources -making it difficult to find and integrate highquality and relevant data.…”

AutophagyNet: high-resolution data source for the analysis of autophagy and its regulation

“…While initially it was thought that autophagy is solely regulated at the protein level, a seminal report in 1999 showed that gene regulatory changes affect autophagy at a transcriptional level [ 65 ]. Moreover, autophagy regulation on the post-transcriptional level by non-coding RNAs has also been described, but less studied [ 66 , 67 ]. Although the knowledge on autophagy regulation has been expanded significantly, data on post-translational and (post-)transcriptional regulations are scattered across many resources – making it difficult to find and integrate high-quality and relevant data.…”

Wdr4 promotes cerebellar development and locomotion through Arhgap17-mediated Rac1 activation

“…These cDNA fragments were then subcloned into pRK5 or pLAS5w.PeGFP with a V5 or Flag tag for transient overexpression or packaging lentiviruses carrying Wdr4, respectively. Lentiviruses were generated as previously described [ 24 ]. Wdr4 mutants were generated using site-directed mutagenesis with the following primers: Wdr4 D166A: 5′- TTTGTGCTTACTGCAG C CCGGGATGAGAAGATC-3′ and 5′-GATCTTCTCATCCCGG G CTGCAGTAAGCACAAA-3′, Wdr4 R172Q: 5′-CGGGATGAGAAGATCC A GGTCAGCTGGGCTGCT-3′ and 5′-AGCAGCCCAGCTGACC T GGATCTTCTCATCCCG-3′.…”